This proposal deals with sarcoplasmic reticulum (SR), a relatively simple membrane which has a vital role in heart and skeletal muscle function. The long-term goal is to characterize this specialized membrane both structurally and functionally in molecular terms. Our major approach in understanding SR has been one of isolation, characterization, dissociation and reconstitution of different types of SR vesicles from skeletal muscle. We have succeeded in purifying and partially characterizing several of the SR proteins. Also, we have defined the conditions which result in the reformation of a functional SR compartment from the solubilized (but not purified) components. We now propose to isolate the Ca2 ion pump - ATPase protein of SR free of phospholipid without irreversible loss of activity. The role of each protein and phospholipid in SR structure and function can then be determined by reconstituting SR vesicles from its purified components. We will study the release of Ca2 ion in different types of native and reconstituted SR vesicles to obtain additional criteria for a functional membrane as well as to gain insight into the poorly understood process of excitation - contraction coupling of skeletal muscle. The reassembly process of solubilized SR protein and phospholipid and the turnover of protein and phospholipid will be examined as a first step toward a study of the biogenesis of SR. The work with SR from skeletal muscle will be extended to the isolation and characterization of SR from heart and diseased muscle. With a comprehensive examination of SR function and assembly will come a better understanding of the action of SR in overall function of normal skeletal and heart muscle as well as diseased muscle.